This project aims at elucidating the pathophysiology of hemolytic anemias secondary to defects in red cell metabolism. We will investigate in detail the glycolytic process of the red cell "in vitro" utilizing newly developed techniques that allow metabolic flow to proceed unimpeded by metabolic block. This is a novel approach to the study of red cell metabolism, that shall permit analysis of glycolysis in detail, in conditions similar to those that exist in the red cell "in vivo". We will investigate in detail the biochemical changes that occur during the process of red cell aging "in vivo". We will examine, through analysis of glycolytic intermediates in age-fractionated red cells, the progressive development of metabolic alterations. We will study in great detail two key enzymes of glycolysis - hexokinase and pyruvate kinase. We will attempt to clarify their molecular structure and to assess the role played by isozymic shifts in the process of molecular aging of enzymes, versus the possibility that this may result from post-translational modifications.